We have conducted two studies on the role of cellular and humoral immune responses in course of HCV infection. Project 1: Hepatitis C virus (HCV) readily establishes chronic infection with exhaustion of HCV-specific T cells and escape from neutralizing antibodies. Spontaneous recovery from chronic infection is rare. Individual cases of HCV clearance have been reported after superinfection with hepatitis A, hepatitis B or hepatitis D viruses, orthotopic liver transplantation, surgery or in the absence of precipitating events but the underlying immunological mechanisms of HCV clearance in these instances have never been studied. Here, we identified an individual who progressed from acute to chronic HCV infection but cleared infection more than 65 weeks after infection without therapy. To investigate the cellular and humoral events underlying this rare phenomenon, we performed a detailed prospective analysis that included IFN-&#947; ELISPOTS, phenotyping of HCV-specific T cells, plasma cytokine estimations, neutralizing antibody assays, and HCV sequencing from prior to infection through more than 2 years of follow-up. In the acute phase of HCV infection we observed a significant increase in plasma cytokines, and a broad HCV-specific T cell and antibody response. However, as typical for HCV, these did not result in viral clearance. Frequency and effector function of HCV-specific T cells decreased during chronic infection and HCV titers stabilized. The unexpected HCV clearance after 65 weeks of chronic infection was associated with reversal of HCV-specific T cell exhaustion, as evidenced by reduced PD-1 expression and improved T cell function, and by appearance of neutralizing antibodies against a conformational epitope. Clearance occurred in the absence of inflammation or superinfection with HBV, CMV, influenza and EBV. Collectively these results demonstrate that T cell exhaustion in chronic HCV infection is reversible and this reversion in conjunction with neutralizing antibodies may clear chronic HCV infection. These findings are relevant for immunotherapy of chronic infections. Project 2: In this project we evaluated cellular immune responses to better define the clinical relevance of persistent recombinant immunoblot assay (RIBA)-indeterminate reactions. The RIBA assay is used to determine the specificity of antibody to hepatitis&#8195;C virus (anti-HCV) and results are recorded as positive, negative, or indeterminate. The interpretation and significance of RIBA-indeterminate reactions are unclear. In collaboration with Dr. Harvey Alter, Department of Transfusion Medicine, Clinical Center, NIH we addressed the clinical relevance of these reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV-positive blood donors. Specifically, we compared HCV-specific T cell responses in 15 RIBA-indeterminate subjects, nine chronic HCV carriers, and eight spontaneously recovered subjects. We found that HCV-specific T cell responses in RIBA-indeterminate subjects were similar to spontaneously recovered subjects and greater than chronic carriers and controls (p&#8195;<&#8195;0.008). The demonstation of HCV-specific T cell responses in RIBA-indeterminate subjects suggest that persistent RIBA-indeterminate reactions represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately disappear leaving no residual serologic evidence of prior HCV infection. These results provide insights into the natural history of hepatitis C virus infection and implications for donor counseling.